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Abstract

The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.

Background

This protocol was developed in order to assess the changes of HIV-1 internalization upon disruption of actin nucleation in human monocyte derived dendritic cells. Following a shRNA screen to identify genes important for HIV-1 transfer from dendritic cells to T cells, we observed that a disruption of actin nucleation leads to a switch from actin rich dendrites to blebs, due to an excess of actomyosin contraction. As a consequence, a decrease of HIV-1 transfer and an increase of HIV-1 internalization due to bleb retraction-driven macropinocytosis were observed. We concluded that effectors of actin nucleation and stabilization were key to maintain HIV-1 on actin-rich dendrites and to limit its endocytosis, for efficient transfer to T lymphocytes (Menager and Littman, 2016).

Proceed to flow cytometry analysis.Note: For flow cytometry, BD high throughput sampler (HTS) can be used to run 96-well plates.

Analysis of HIV-1 capture by Quantitative PCR (QPCR)
Real time quantitative PCR (qPCR) was performed after reverse transcription using a Roche LightCycler 480 with Roche 480 SYBR Green I Master reagent according to manufacturer specifications. The relative abundance of HIV is calculated based on the amount of GFP mRNA (the virus encodes GFP) using a standard curve and normalized using GAPDH as a control.

Using a confocal microscope, analyze for each cell the intensity of internalized GFP or FITC, which represent, respectively, captured HIV-1 particles and dextran molecules. To do so, acquire 400 nm Z-stacks and determine the HIV-1 and dextran molecules. Quantification of HIV-1 and dextran uptake will be discussed in the Data analysis section. (Figures 1 and 2)

Figure 1. Example of untransduced and untreated MDDCs loaded for 4 h with 100 μg/ml of 10 kDa FITC-labeled dextran molecules. Cells on coverslips can be seen on the left panel (bright-field). On the right panel, one Z-stack section of 400 nm is displayed where nuclei of cells were stained with DAPI (blue) and molecules of dextran can be visualized in green.

Figure 2. Example of HIV-1 internalization by confocal microscopy. MDDCs untransduced (top panel) or transduced with a shRNA against DNM2 (bottom panel) loaded for 4 h with 50 ng of HIV-1 (green dots). Bright-field can be seen on the left panel. On the right panel, one Z-stack section of 400 nm is displayed where nuclei of cells were stained with DAPI (blue), actin filaments in red, after phalloidin staining and captured HIV-1 in green.

Data analysis

Analysis of HIV-1 or dextran capture by flow cytometry (Figure 3)

First, the gate must be set on live cells, by excluding cells that have incorporated the live/dead stain. It is important that MDDCs are well differentiated, as assessed by high expression of DC-SIGN and low expression of CD14. (Figure 3A)

Monocytes are usually CD14 high and DC-SIGN low.

The amount of HIV-1 or dextran internalized can be calculated by comparing the mean fluorescence intensity (MFI) to the negative control and determining the ratio of internalization between samples (Figure 3B).

It is recommended that HIV and dextran capture be assessed by using different dilutions and different times of incubation, in order to draw a representative and more accurate curve.

Each sample is usually analyzed in triplicate and the experiment is performed with cells coming from at least 3 independent healthy blood donors.

Figure 3. Analysis of HIV-1 and dextran capture by flow cytometry. A. Gating strategy to analyse MDDCs: left panel, gating on MDDCs based on size (FSC) and granularity (SSC); right panel gating on alive differentiated MDDCs (DC-SIGN+; dapi-). B. Example of histograms generated after gating on MDDCs loaded with HIV-1 (top panel) or dextran (lower panel). In grey, an isotype control for P24 is used to assess the amount of HIV-1 captured by MDDCs transduced with scramble shRNA vs. TSPAN7 shRNA. For dextran internalization, the grey histograms represent a negative control where cells are staying at 4 °C to prevent internalization.

Analysis of HIV-1 capture by Quantitative PCR (QPCR) (Figure 4)

After reverse transcription, the amount of HIV-1 RNA was detected by quantitative PCR with primers for GFP (encoded inside the HIV genome) and normalized to GAPDH.

To assess the quantity in the most accurate way possible, every experiment should include a standard curve using the GFP primers and also the GAPDH primers.

Then the standard curve is used to determine the quantity of HIV internalized in each sample and compare that to the negative controls (no virus or capture experiment performed at 4 °C).

Normalized HIV-1 quantity can be compared between different samples to assess the increase or the decrease in HIV capture (Figure 4).

Due to experimental variations that could exist between different blood donors, it is preferable not to compare samples coming from different donors, but seek consistency between donors in terms of trends. Experiments should be done in triplicate and in at least five different blood donors.

Figure 4. Analysis of HIV-1 capture by quantitative PCR. Example of histograms after HIV capture experiments and analysis by quantitative PCR. MDDCs transduced with scramble shRNA, empty vectors or shRNA against TSPAN7 were loaded with HIV-GFP for 4 or 24 h. Quantity of HIV-1 captured is analysed by PCR against GFP, and compared to the quantity captured after scramble shRNA (set arbitrarily to a value of 1).

For each sample, we recommend to calculate the concentration of dextran for at least 100 cells and to repeat the experiment with at least 3 different human blood donors. Unpaired t-test can be used to determine statistical significance.

Regarding quantification of HIV-1 internalization, 400 nm Z-stacks are acquired using a confocal microscope with an average of 20 Z-stacks per cell.

Every HIV-1 particle (green dot) can be manually counted as internalized based on its localization compared to the cortical actin barrier (phalloidin staining).

Based on electron microscopy data, an average of 5 HIV viral particles was estimated to be present per HIV-1 aggregate.

For each cell, the total of HIV-1 can thus be calculated and an average of at least 100 cells per sample is analyzed. Unpaired t-test can be used to determine statistical significance.

Figure 5. Analysis of dextran capture by confocal microscopy. MDDCs transduced with a scramble shRNA or shRNA against TSPAN7 were cultured for 4 h with 10 kDa (left panel) or 70 kDa molecules of dextran (right panel). The concentration of dextran internalized by each MDDC was then determined as explained above. Briefly, concentration is the integrated density of dextran staining divided by volume and is displayed with arbitrary units on the y axis. For each condition, each dot represents a different cell and the average is displayed ± SEM.

Notes

In general, when working with human primary cells, you need to make sure to have internal controls in order to be able to normalize the data and to compare data coming from different blood donors. Variation can also be observed from donor to donor and increasing the number of different human blood donors usually helps to have better analysis of the data.

We recommend to always check the status of maturation of MDDCs. Experiments as described in this protocol are performed using immature MDDCs. Once mature, the results obtained in terms of HIV-1 capture and dextran internalization are different. Maturation status can be determined by looking at CD86 and CD80 molecules by flow cytometry. Immature MDDCs should be CD80 and CD86 low.

All 3 techniques measure HIV-1 internalization by MDDCs. Combining 3 different methods is increasing the chances to detect even small differences in capture and internalization and is increasing the robustness of the results. To detect changes in HIV-1 internalization, flow cytometry is maybe not the best method as it doesn’t seem to be as sensitive as QPCR or confocal microscopy to detect small amount of HIV internalization. Quantitative PCR is maybe the fastest and easiest method but is not allowing a direct comparison of HIV-1 and dextran internalization and any artefactual contamination could potentially biased the data. The most demanding technique is probably the confocal microscopy but allows the most rigorous comparison of both HIV-1 and dextran internalization between different samples.

We thank Alice F. Liang, Michael Cammer and the NYULMC OCS Microscopy Core for the service provided for light microscopy; Wendy Lin for technical help; and Jarrod Johnson, Nicolas Manel, for their critical advice. This work was supported by fellowships from EMBO, the Cancer Research Institute, and the Philippe Foundation (M.M.M.); by the Howard Hughes Medical Institute (D.R.L.) and Helen and Martin Kimmel Center for Biology and Medicine (D.R.L.); and by grants from the National Institutes of Health (R21AI084633) (D.R.L.) and NCRR (S10RR023704-01A1).